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GATA2(-/-) human ESCs undergo attenuated endothelial to hematopoietic transition and thereafter granulocyte commitment.

Huang K, Du J, Ma N, Liu J, Wu P, Dong X, Meng M, Wang W, Chen X, Shi X, Chen Q, Yang Z, Chen S, Zhang J, Li Y, Li W, Zheng Y, Cai J, Li P, Sun X, Wang J, Pei D, Pan G - Cell Regen (Lond) (2015)

Bottom Line: Our results demonstrated that GATA2 (-/-) hESCs displayed attenuated generation of CD34(+)CD43(+) hematopoietic progenitor cells (HPCs), due to the impairment of endothelial to hematopoietic transition (EHT).Interestingly, GATA2 (-/-) hESCs retained the potential to generate erythroblasts and macrophages, but never granulocytes.Furthermore, we found that GATA2 (-/-) hESCs restored the granulocyte potential in the presence of Notch signaling.

View Article: PubMed Central - PubMed

Affiliation: CAS Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China ; Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China.

ABSTRACT

Background: Hematopoiesis is a progressive process collectively controlled by an elaborate network of transcription factors (TFs). Among these TFs, GATA2 has been implicated to be critical for regulating multiple steps of hematopoiesis in mouse models. However, whether similar function of GATA2 is conserved in human hematopoiesis, especially during early embryonic development stage, is largely unknown.

Results: To examine the role of GATA2 in human background, we generated homozygous GATA2 knockout human embryonic stem cells (GATA2 (-/-) hESCs) and analyzed their blood differentiation potential. Our results demonstrated that GATA2 (-/-) hESCs displayed attenuated generation of CD34(+)CD43(+) hematopoietic progenitor cells (HPCs), due to the impairment of endothelial to hematopoietic transition (EHT). Interestingly, GATA2 (-/-) hESCs retained the potential to generate erythroblasts and macrophages, but never granulocytes. We further identified that SPI1 downregulation was partially responsible for the defects of GATA2 (-/-) hESCs in generation of CD34(+)CD43(+) HPCs and granulocytes. Furthermore, we found that GATA2 (-/-) hESCs restored the granulocyte potential in the presence of Notch signaling.

Conclusion: Our findings revealed the essential roles of GATA2 in EHT and granulocyte development through regulating SPI1, and uncovered a role of Notch signaling in granulocyte generation during hematopoiesis modeled by human ESCs.

No MeSH data available.


Related in: MedlinePlus

Characterization of subtype blood lineages from H1 or H1-GATA2−/− derived HPCs. a CFU potential cells from H1 or H1-GATA2−/− were restricted within CD34+CD43+ subpopulations. EC endothelial cells, MC mesenchymal cells. b Characterization of erythrocytes from H1 or H1-GATA2−/−. From left to right: phase-contrast photographs of BFU and CFU-E, FACS analysis of CD235a and CD71a expression on H1 and H1-GATA2−/− derived erythrocytes, and cytospin of H1 and H1-GATA2−/− derived erythrocytes. c Globin analysis of erythrocytes by RT-qPCR. The results showed the mean + SEM of one single experiment with three replicates, representative of three independent experiments. d Analysis of expression of GATA1, GATA2, and GATA3 in H1 or H1-GATA2−/− derived erythrocytes. The results showed the mean + SEM of one single experiment with three replicates, representative of three independent experiments. e Characterization of myeloid cells from H1 or H1-GATA2−/−. Left: morphologies of indicated CFU colonies; middle: FACS analysis of indicated markers; right: cytospin photographs of indicated colonies. f FACS analysis of CD86 and CD14 expression in H1 and H1-GATA2−/− derived myeloid CFU. E erythrocyte, G granulocyte, M macrophage, GM G and M, Mix G, E, and M
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Fig3: Characterization of subtype blood lineages from H1 or H1-GATA2−/− derived HPCs. a CFU potential cells from H1 or H1-GATA2−/− were restricted within CD34+CD43+ subpopulations. EC endothelial cells, MC mesenchymal cells. b Characterization of erythrocytes from H1 or H1-GATA2−/−. From left to right: phase-contrast photographs of BFU and CFU-E, FACS analysis of CD235a and CD71a expression on H1 and H1-GATA2−/− derived erythrocytes, and cytospin of H1 and H1-GATA2−/− derived erythrocytes. c Globin analysis of erythrocytes by RT-qPCR. The results showed the mean + SEM of one single experiment with three replicates, representative of three independent experiments. d Analysis of expression of GATA1, GATA2, and GATA3 in H1 or H1-GATA2−/− derived erythrocytes. The results showed the mean + SEM of one single experiment with three replicates, representative of three independent experiments. e Characterization of myeloid cells from H1 or H1-GATA2−/−. Left: morphologies of indicated CFU colonies; middle: FACS analysis of indicated markers; right: cytospin photographs of indicated colonies. f FACS analysis of CD86 and CD14 expression in H1 and H1-GATA2−/− derived myeloid CFU. E erythrocyte, G granulocyte, M macrophage, GM G and M, Mix G, E, and M

Mentions: As shown in Fig. 2a, although we could easily detect the major colony-forming cell (CFC) types including erythroid (CFC-E or BFU) and myeloid (granulocytes and macrophages, CFU-Mix) from WT hESCs upon co-culturing with OP9, we only observed erythroid and macrophage CFCs from GATA2−/− CD34+ cells. We never detected granulocytes (CFU-G) from GATA2−/− CD34+ cells. Consistent with previous findings, we showed that the potential cells were restricted within the population of CD34+CD43+ (Fig. 3a) [23]. These data indicated that GATA2 might particularly target and regulate granulocyte specification in human ESC-based hematopoiesis.Fig. 3


GATA2(-/-) human ESCs undergo attenuated endothelial to hematopoietic transition and thereafter granulocyte commitment.

Huang K, Du J, Ma N, Liu J, Wu P, Dong X, Meng M, Wang W, Chen X, Shi X, Chen Q, Yang Z, Chen S, Zhang J, Li Y, Li W, Zheng Y, Cai J, Li P, Sun X, Wang J, Pei D, Pan G - Cell Regen (Lond) (2015)

Characterization of subtype blood lineages from H1 or H1-GATA2−/− derived HPCs. a CFU potential cells from H1 or H1-GATA2−/− were restricted within CD34+CD43+ subpopulations. EC endothelial cells, MC mesenchymal cells. b Characterization of erythrocytes from H1 or H1-GATA2−/−. From left to right: phase-contrast photographs of BFU and CFU-E, FACS analysis of CD235a and CD71a expression on H1 and H1-GATA2−/− derived erythrocytes, and cytospin of H1 and H1-GATA2−/− derived erythrocytes. c Globin analysis of erythrocytes by RT-qPCR. The results showed the mean + SEM of one single experiment with three replicates, representative of three independent experiments. d Analysis of expression of GATA1, GATA2, and GATA3 in H1 or H1-GATA2−/− derived erythrocytes. The results showed the mean + SEM of one single experiment with three replicates, representative of three independent experiments. e Characterization of myeloid cells from H1 or H1-GATA2−/−. Left: morphologies of indicated CFU colonies; middle: FACS analysis of indicated markers; right: cytospin photographs of indicated colonies. f FACS analysis of CD86 and CD14 expression in H1 and H1-GATA2−/− derived myeloid CFU. E erythrocyte, G granulocyte, M macrophage, GM G and M, Mix G, E, and M
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Fig3: Characterization of subtype blood lineages from H1 or H1-GATA2−/− derived HPCs. a CFU potential cells from H1 or H1-GATA2−/− were restricted within CD34+CD43+ subpopulations. EC endothelial cells, MC mesenchymal cells. b Characterization of erythrocytes from H1 or H1-GATA2−/−. From left to right: phase-contrast photographs of BFU and CFU-E, FACS analysis of CD235a and CD71a expression on H1 and H1-GATA2−/− derived erythrocytes, and cytospin of H1 and H1-GATA2−/− derived erythrocytes. c Globin analysis of erythrocytes by RT-qPCR. The results showed the mean + SEM of one single experiment with three replicates, representative of three independent experiments. d Analysis of expression of GATA1, GATA2, and GATA3 in H1 or H1-GATA2−/− derived erythrocytes. The results showed the mean + SEM of one single experiment with three replicates, representative of three independent experiments. e Characterization of myeloid cells from H1 or H1-GATA2−/−. Left: morphologies of indicated CFU colonies; middle: FACS analysis of indicated markers; right: cytospin photographs of indicated colonies. f FACS analysis of CD86 and CD14 expression in H1 and H1-GATA2−/− derived myeloid CFU. E erythrocyte, G granulocyte, M macrophage, GM G and M, Mix G, E, and M
Mentions: As shown in Fig. 2a, although we could easily detect the major colony-forming cell (CFC) types including erythroid (CFC-E or BFU) and myeloid (granulocytes and macrophages, CFU-Mix) from WT hESCs upon co-culturing with OP9, we only observed erythroid and macrophage CFCs from GATA2−/− CD34+ cells. We never detected granulocytes (CFU-G) from GATA2−/− CD34+ cells. Consistent with previous findings, we showed that the potential cells were restricted within the population of CD34+CD43+ (Fig. 3a) [23]. These data indicated that GATA2 might particularly target and regulate granulocyte specification in human ESC-based hematopoiesis.Fig. 3

Bottom Line: Our results demonstrated that GATA2 (-/-) hESCs displayed attenuated generation of CD34(+)CD43(+) hematopoietic progenitor cells (HPCs), due to the impairment of endothelial to hematopoietic transition (EHT).Interestingly, GATA2 (-/-) hESCs retained the potential to generate erythroblasts and macrophages, but never granulocytes.Furthermore, we found that GATA2 (-/-) hESCs restored the granulocyte potential in the presence of Notch signaling.

View Article: PubMed Central - PubMed

Affiliation: CAS Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China ; Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China.

ABSTRACT

Background: Hematopoiesis is a progressive process collectively controlled by an elaborate network of transcription factors (TFs). Among these TFs, GATA2 has been implicated to be critical for regulating multiple steps of hematopoiesis in mouse models. However, whether similar function of GATA2 is conserved in human hematopoiesis, especially during early embryonic development stage, is largely unknown.

Results: To examine the role of GATA2 in human background, we generated homozygous GATA2 knockout human embryonic stem cells (GATA2 (-/-) hESCs) and analyzed their blood differentiation potential. Our results demonstrated that GATA2 (-/-) hESCs displayed attenuated generation of CD34(+)CD43(+) hematopoietic progenitor cells (HPCs), due to the impairment of endothelial to hematopoietic transition (EHT). Interestingly, GATA2 (-/-) hESCs retained the potential to generate erythroblasts and macrophages, but never granulocytes. We further identified that SPI1 downregulation was partially responsible for the defects of GATA2 (-/-) hESCs in generation of CD34(+)CD43(+) HPCs and granulocytes. Furthermore, we found that GATA2 (-/-) hESCs restored the granulocyte potential in the presence of Notch signaling.

Conclusion: Our findings revealed the essential roles of GATA2 in EHT and granulocyte development through regulating SPI1, and uncovered a role of Notch signaling in granulocyte generation during hematopoiesis modeled by human ESCs.

No MeSH data available.


Related in: MedlinePlus